Class-switch DNA recombination (CSR) and somatic hypermutation (SHM), which require AID, and plasma cell differentiation, which requires Blimp-1, are critical for the generation of class-switched and hypermutated (mature) antibody and autoantibody responses. We showed here that the histone deacetylase (HDAC) inhibitors (HDI) valproic acid (VPA) and butyrate upregulated miR-155, miR-181b and miR-361, which silenced AICDA/Aicda (AID) mRNA, and miR-23b, miR-30a and miR-125b, which silenced PRDM1/Prdm1 (Blimp-1) mRNA, in human and mouse B cells. This led to downregulation of AID, Blimp-1 and Xbp-1 expression, thereby dampening CSR, SHM and plasma cell differentiation without altering B cell viability or proliferation. The selectivity of HDI-mediated silencing of AICDA/Aicda and PRDM1/Prdm1 was emphasized by unchanged expression of HoxC4 and Irf4 (important inducers/modulators of AICDA/Aicda), Rev1 and Ung (central elements for CSR/SHM), and Bcl6, Bach2 or Pax5 (repressors of PRDM1/Prdm1 expression), as well as unchanged expression of miR-19a/b, miR-20a and miR-25, which are not known to regulate AICDA/Aicda or PRDM1/Prdm1. Through these B cell intrinsic epigenetic mechanisms, VPA blunted class-switched and hypermutated T-dependent and T-independent antibody responses in C57BL/6 mice. In addition, it decreased class-switched and hypermutated autoantibodies, ameliorated disease and extended survival in lupus MRL/Faslpr/lpr mice. Our findings outline epigenetic mechanisms that modulate expression of an enzyme (AID) and transcription factors (Blimp-1 and Xbp-1) that critical to the B cell differentiation processes that underpin antibody and autoantibody responses. They also provide therapeutics proof-of-principle in autoantibody-mediated autoimmunity.

Summary B cell depletion potently reduces episodes of inflammatory demyelination in multiple sclerosis (MS), predominantly through loss of innate rather than adaptive immunity. However, molecular mechanisms controlling innate versus adaptive B cell function are poorly understood. N -glycan branching, via interactions with galectins, controls endocytosis and signaling of cell surface receptors to control cell function. Here we report that N -glycan branching in B cells dose dependently reduces pro-inflammatory innate responses by titrating decreases in Toll-like receptor-4 (TLR4) and TLR2 surface expression via endocytosis. In contrast, a minimal level of N -glycan branching maximizes surface retention of the B cell receptor (BCR) and the CD19 co-receptor to promote adaptive immunity. Branched N -glycans inhibit antigen presentation by B cells to reduce T helper cell-17 (T H 17)/T H 1 differentiation and inflammatory demyelination in mice. Thus, N -glycan branching negatively regulates B cell innate function while promoting/maintaining adaptive immunity via BCR, providing an attractive therapeutic target for MS.

Osteosarcoma is the most common primary malignant neoplasm of bone and typically occurs in children and young adults. As a highly metastatic malignancy, 15–20% of osteosarcoma patients are diagnosed after the tumor has already metastasized (typically to the lungs), which translates to 5-year survival rates of <40%. Here, we tested the effect of the cyclin-dependent kinase (CDK) inhibitor flavopiridol (alvocidib) in U2OS, SaOS-2, SJSA-1, and 143B osteosarcoma tumor cells in vitro and in vivo. Our results show that flavopiridol can drastically decrease survival in these osteosarcoma cell lines at nanomolar concentrations and induce mitotic catastrophe in p53-null osteosarcomas. We also performed transcriptome analysis (RNA-seq) of flavopiridol-treated osteosarcoma cells, which revealed significant changes in genes coding for proteins involved in cell-cell and cell-matrix adhesions, including cadherin 3 (CDH3) and 4 (CDH4). These transcriptional changes translated to a striking reduction in the ability of osteosarcoma cells to migrate and invade in vitro. Further, in vivo assessment of the effects of flavopiridol on osteosarcoma metastasis resulted in a significant reduction in the number of lung metastases in mice treated with flavopiridol at concentrations that are physiologically tolerable. This study suggests that flavopiridol, likely in combination with other cytotoxic chemotherapeutic agents, may be a promising drug for the treatment of osteosarcoma.

No abstract

Allogeneic off-the-shelf cell therapies offer distinct advantages over conventional autologous cell therapies in terms of scaled manufacturing, on-demand availability and optimization of cellular starting material. A unique consideration in the use of allogeneic cell therapies is the potential for immune cell-mediated recognition of the allogeneic cell product by the patient's immune system. CAR T-cell therapies are commonly combined with conditioning chemotherapies that suppress a patient's immune system, creating a suitable window of activity to elicit clinical response. However, protracted lympho-conditioning also affects immune reconstitution and can negatively impact the rate of infection. Alternative approaches to prevent allorejection may therefore help to enhance the efficacy of the therapy while preserving the immune system of the patient. Elimination of cell-surface human leukocyte antigen (HLA) molecule expression by genetic knockout (KO) has long been known to abrogate T-cell reactivity. However, loss of class I HLA elicits NK cell-mediated recognition and clearance, and therefore must be combined with other immune-modulating strategies to limit host NK cell reactivity. Allogeneic models combining class I HLA deletion with NK cell inhibitory molecules, such as HLA-E and CD47, have been shown to abrogate NK cell reactivity in mouse models. However, HLA-E is the canonical activator of NKG2C, a dominant activating receptor found on human NK cells. Likewise, the expression of signal regulatory protein alpha (SIRPα), the major interactor for CD47, is mostly restricted to macrophages and dendritic cells and not human NK cells, and the observed effects of this immune-modulating strategy in the mouse system may only offer partial or incomplete immune evasion in the human system. In this study, we provide details of a bona fide off-the-shelf strategy where iPSC-derived NK (iNK) cell therapy is multiplexed engineered with a novel combination of immune-evasion modalities; beta 2 microgobulin (B2M) KO to prevent CD8 T-cell mediated rejection; class II transactivator (CIITA) KO to prevent CD4 T-cell mediated rejection; and CD38 KO to enable combination with anti-CD38 mAbs, which can be administered to deplete host alloreactive lymphocytes, including both NK and T cells. In vitro mixed lymphocyte reaction (MLR) data demonstrated that upon co-culture with allogeneic PBMCs, B2M KO iNK cells stimulated less T-cell activation than their B2M sufficient counterparts as evidenced by reduced CD38, 41BB, and CD25 levels on T cells. Additionally, B2M KO iNK cells impaired T-cell expansion over the duration of co-culture, resulting in a 50% decrease in expansion at the peak of the control response. However, B2M KO iNK cells were depleted over time, suggesting activation of an NK cell "missing self" response by the peripheral blood NK (pbNK) cells. In contrast, when the assay was performed in the presence of anti-CD38 mAb, depletion of B2M KO iNK cells was blocked, and instead B2M KO iNK cell numbers increased by 3.5-fold, comparable to the iNK cell numbers found in the control arm (cultured without allogeneic PBMCs). Interestingly, pbNK cell numbers decreased, while T-cell activation and expansion remained lower than in B2M-sufficient MLR cultures. Furthermore, when B2M KO iNK cells were cocultured with tumor cells and anti-CD38 mAb in vitro, ADCC was comparable to the B2M sufficient cells, indicating uncompromised effector function. Finally, in vivo studies suggested that co-administration of anti-CD38 mAbs can significantly enhance the persistence of B2M KO iNK cells in the presence of allogeneic pbNK cells as seen in the spleen and bone marrow (Figure 1). Together these data demonstrate that the combination of triple-gene knockout of CD38, B2M and CIITA with a CD38-targeting mAb is an effective strategy to avoid host immune rejection, and highlights the potential advantages of multiplexed engineered iPSCs to facilitate large-scale manufacture of complex engineered, off-the-shelf cellular therapies. Figure 1 Figure 1. Disclosures Williams: Fate Therapeutics: Current Employment. Malmberg: Merck: Research Funding; Vycellix: Consultancy; Fate Therapeutics: Consultancy, Research Funding. Lee: Fate Therapeutics, Inc.: Current Employment. Bjordahl: Fate Therapeutics: Current Employment. Valamehr: Fate Therapeutics, Inc.: Current Employment.

Chimeric antigen receptor (CAR) T-cell therapies have revolutionized the treatment of hematologic malignancies, however, logistical complexities associated with patient-specific CAR T-cell therapies often limit broad accessibility to patients. Many of these challenges can be overcome with an allogeneic cellular product that is available off-the-shelf, and overcoming immune cell-mediated rejection of allogeneic cell therapy is an area of significant research. Conditioning chemotherapies, which are commonly administered with CAR T-cell therapy, suppress a patient's immune system and may create a suitable window of activity for allogeneic cell therapies to elicit clinical response. However, protracted lympho-conditioning has been associated with poor immune reconstitution and increased susceptibility to opportunistic infections. Deletion of human leukocyte antigen (HLA) surface expression is known to abrogate T-cell alloreactivity, but deletion of class I HLA must be combined with other immune-modulating strategies to avoid NK cell-mediated recognition. To this end, allogeneic models combining class I HLA deletion with NK cell inhibitory molecules, such as HLA-E and CD47, have been shown to abrogate NK cell reactivity in mouse models. However, since HLA-E is the canonical activator of NKG2C, a dominant activating receptor found on human NK cells, and since the ligand for CD47, SIRPα, is known to be expressed on macrophages and dendritic cells and not on human NK cells, the observed effects of these immune-modulating strategies may not translate into the patient-treatment setting. In assessment of their protective effects in a defined human system, we found that HLA-E or CD47 overexpression on class I HLA null human cells offer only partial protection in evading various human NK cell compartments. We found that class I HLA-null K562 cells engineered to over-express CD47 were ineffective in inhibiting NK cells (0 to 7% inhibition). Separately, K562 cells engineered to over-express HLA-E, while effective in inhibiting NKG2A+ NK cells (90.2% +/- 3.7% inhibition), were unable to completely inhibit CD56 dim NK cells (33.2% +/- 29.6% inhibition) and not only failed, but instead activated, NKG2C+ NK cells (167% +/- 69% activation). Our data highlight the limitations of engineered CD47 and HLA-E modalities in suppressing broad populations of NK cells in clinically relevant settings. We therefore evaluated expression of the alloimmune defense receptor (ADR) that uniquely targets alloreactive immune cells (Mo et al. Nat Biotechnol 2021). We have shown that the expression of ADR has the potential to evade host immune cells without the need for further genetic editing such as class I HLA deletion. To determine its applicability for off-the-shelf cell therapy, ADR expression was engineered into induced pluripotent stem cells (iPSCs) and iPSC-derived CAR-NK (CAR-iNK) cells were generated. CAR-iNK cells carrying the ADR modality (ADR+ CAR-iNK cells) showed normal patterns of differentiation (&gt;99% CD56+ with co-expression of NK cell receptors such as NKG2D, NKp30 and NKp46), suggesting that ADR expression did not disrupt hematopoiesis or the expansion of iNK cells. Additionally, ADR+ and ADR-negative CAR-iNK cells produced similar cytotoxicity against tumor cells. We next tested the ability of ADR to provide resistance to alloimmune rejection by coculturing ADR+ CAR-iNK cells with allogeneic pBMCs in a mixed lymphocyte reaction (MLR) assay. Notably, ADR+ CAR-iNK cells maintained durable persistence throughout the entire duration of the MLR assay and suppressed the expansion of alloreactive T- and NK-cells in comparison to the control arm (Figure 1). Collectively, initial preclinical studies suggest that ADR-modified CAR-iNK cells resist host immune cell rejection, while eliciting a durable anti-tumor response. Our preliminary data show evidence toward a promising off-the-shelf solution for elimination of broad pools of alloreactive T- and NK- effector cells in the clinical setting without the need for lympho-depleting conditioning or genetic editing strategies. Figure 1 Figure 1. Disclosures Williams: Fate Therapeutics: Current Employment. Abujarour: Fate Therapeutics, Inc.: Current Employment. Lee: Fate Therapeutics, Inc.: Current Employment. Malmberg: Merck: Research Funding; Vycellix: Consultancy; Fate Therapeutics: Consultancy, Research Funding. Mamonkin: Beam Therapeutics: Other: Licensing payments; Fate Therapeutics: Other: Licensing payments; Allogene Therapeutics: Consultancy, Other: Licensing payments; Xenetic Biosciences: Consultancy, Membership on an entity's Board of Directors or advisory committees. Bjordahl: Fate Therapeutics: Current Employment. Valamehr: Fate Therapeutics, Inc.: Current Employment.

Aging is associated with a decline in immune function, increasing the frequency and severity of infection in the elderly. We have previously shown that Asparagine (N) – linked protein glycosylation is a critical negative regulator of T cell immunity in both mice and humans. Here we report N-glycan branching in T cells increases with age, leading to T cell hypo-activity and increased susceptibility to infection. Reducing N-glycan branching rejuvenates T cell activation, proliferation and pro-inflammatory TH17 over anti-inflammatory Treg differentiation in aged T cells. Susceptibility of aged mice to Salmonellae typhimurium invasion/dissemination was reduced by lowering N-glycan branching. A critical metabolic precursor of N-glycosylation and branching is N-acetylglucosamine (GlcNAc), a common amino sugar that is part of the regular human diet. GlcNAc is endogenous to human serum, increases with age and correlates with N-glycan branching in T cells. T cell specific deletion of N-acetylglucosamine kinase, thereby blocking salvage of GlcNAc into N-glycans, reduces branching in aged T cells and rejuvenates T cell activation. Collectively, our results suggest that age-dependent increases in N-glycan branching, via increased supply of GlcNAc, impairs T cell immunity in the elderly. Therapeutic inhibition of N-glycan branching may provide a novel mechanism to rejuvenate T cell responses and reduce infection risk in the elderly.

No abstract